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Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation.

Besseyrias V, Fiorini E, Strobl LJ, Zimber-Strobl U, Dumortier A, Koch U, Arcangeli ML, Ezine S, Macdonald HR, Radtke F - J. Exp. Med. (2007)

Bottom Line: However, N2-DL1-mediated signaling does not allow further T cell maturation beyond the CD25(+) stage due to a lack of T cell receptor beta expression.Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak.Collectively, our results establish a hierarchy of Notch-Delta interactions in which N1-DL4 exhibits the greatest capacity to induce and support T cell development.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland.

ABSTRACT
Notch1 (N1) receptor signaling is essential and sufficient for T cell development, and recently developed in vitro culture systems point to members of the Delta family as being the physiological N1 ligands. We explored the ability of Delta1 (DL1) and DL4 to induce T cell lineage commitment and/or maturation in vitro and in vivo from bone marrow (BM) precursors conditionally gene targeted for N1 and/or N2. In vitro DL1 can trigger T cell lineage commitment via either N1 or N2. N1- or N2-mediated T cell lineage commitment can also occur in the spleen after short-term BM transplantation. However, N2-DL1-mediated signaling does not allow further T cell maturation beyond the CD25(+) stage due to a lack of T cell receptor beta expression. In contrast to DL1, DL4 induces and supports T cell commitment and maturation in vitro and in vivo exclusively via specific interaction with N1. Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak. Interestingly, preferential N1-DL4 binding reflects reduced dependence of this interaction on Lunatic fringe, a glycosyl transferase that generally enhances the avidity of Notch receptors for Delta ligands. Collectively, our results establish a hierarchy of Notch-Delta interactions in which N1-DL4 exhibits the greatest capacity to induce and support T cell development.

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Binding of DL1- and DL4-IgG fusion proteins to N1 and N2. (A) Semiquantitative RT-PCR for the Lfng gene. cDNAs were prepared from DN thymocytes and 293T cells. Transcripts were analyzed by semiquantitative RT-PCR of fivefold dilutions of the cDNA. The cDNA input was normalized according to the expression of the control HPRT gene. 293T cells were transiently transfected with N1- (B) or N2-EGFP (C) together with or without Lfng expression vectors and stained 48 h after transfection with either human IgG1 isotype control or DL1- or DL4-IgG fusion proteins. Data are representative FACS profiles of four independent experiments. Extremely high EGFP-expressing cells were gated out as the fusion proteins were trapped inside the cells.
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fig7: Binding of DL1- and DL4-IgG fusion proteins to N1 and N2. (A) Semiquantitative RT-PCR for the Lfng gene. cDNAs were prepared from DN thymocytes and 293T cells. Transcripts were analyzed by semiquantitative RT-PCR of fivefold dilutions of the cDNA. The cDNA input was normalized according to the expression of the control HPRT gene. 293T cells were transiently transfected with N1- (B) or N2-EGFP (C) together with or without Lfng expression vectors and stained 48 h after transfection with either human IgG1 isotype control or DL1- or DL4-IgG fusion proteins. Data are representative FACS profiles of four independent experiments. Extremely high EGFP-expressing cells were gated out as the fusion proteins were trapped inside the cells.

Mentions: Because the binding assays were performed with WT thymocytes, we were unable to distinguish binding of DL4-IgG to the N1 and/or N2 receptor. Our results presented in Fig. 5 show that DL4, in contrast to DL1, cannot induce T lineage commitment via N2. This result is compatible with two hypotheses: either DL4 cannot bind efficiently to N2 or, alternatively, DL4 can bind N2 but cannot transmit a signal via the N2 receptor. To distinguish between these two possibilities, we examined the binding efficiency of DL1- and DL4-IgG fusion proteins to 293T cells transiently expressing either N1- or N2–enhanced GFP (EGFP) fusion proteins. As shown in Fig. 7 (B and C, top), DL4-IgG fusion proteins bind N1 very efficiently, whereas binding to the N2 receptor is not detectable above background. Interestingly, DL1-IgG fusion proteins bind N1 weakly but do not bind N2 above levels of the IgG isotype control.


Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation.

Besseyrias V, Fiorini E, Strobl LJ, Zimber-Strobl U, Dumortier A, Koch U, Arcangeli ML, Ezine S, Macdonald HR, Radtke F - J. Exp. Med. (2007)

Binding of DL1- and DL4-IgG fusion proteins to N1 and N2. (A) Semiquantitative RT-PCR for the Lfng gene. cDNAs were prepared from DN thymocytes and 293T cells. Transcripts were analyzed by semiquantitative RT-PCR of fivefold dilutions of the cDNA. The cDNA input was normalized according to the expression of the control HPRT gene. 293T cells were transiently transfected with N1- (B) or N2-EGFP (C) together with or without Lfng expression vectors and stained 48 h after transfection with either human IgG1 isotype control or DL1- or DL4-IgG fusion proteins. Data are representative FACS profiles of four independent experiments. Extremely high EGFP-expressing cells were gated out as the fusion proteins were trapped inside the cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2118717&req=5

fig7: Binding of DL1- and DL4-IgG fusion proteins to N1 and N2. (A) Semiquantitative RT-PCR for the Lfng gene. cDNAs were prepared from DN thymocytes and 293T cells. Transcripts were analyzed by semiquantitative RT-PCR of fivefold dilutions of the cDNA. The cDNA input was normalized according to the expression of the control HPRT gene. 293T cells were transiently transfected with N1- (B) or N2-EGFP (C) together with or without Lfng expression vectors and stained 48 h after transfection with either human IgG1 isotype control or DL1- or DL4-IgG fusion proteins. Data are representative FACS profiles of four independent experiments. Extremely high EGFP-expressing cells were gated out as the fusion proteins were trapped inside the cells.
Mentions: Because the binding assays were performed with WT thymocytes, we were unable to distinguish binding of DL4-IgG to the N1 and/or N2 receptor. Our results presented in Fig. 5 show that DL4, in contrast to DL1, cannot induce T lineage commitment via N2. This result is compatible with two hypotheses: either DL4 cannot bind efficiently to N2 or, alternatively, DL4 can bind N2 but cannot transmit a signal via the N2 receptor. To distinguish between these two possibilities, we examined the binding efficiency of DL1- and DL4-IgG fusion proteins to 293T cells transiently expressing either N1- or N2–enhanced GFP (EGFP) fusion proteins. As shown in Fig. 7 (B and C, top), DL4-IgG fusion proteins bind N1 very efficiently, whereas binding to the N2 receptor is not detectable above background. Interestingly, DL1-IgG fusion proteins bind N1 weakly but do not bind N2 above levels of the IgG isotype control.

Bottom Line: However, N2-DL1-mediated signaling does not allow further T cell maturation beyond the CD25(+) stage due to a lack of T cell receptor beta expression.Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak.Collectively, our results establish a hierarchy of Notch-Delta interactions in which N1-DL4 exhibits the greatest capacity to induce and support T cell development.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland.

ABSTRACT
Notch1 (N1) receptor signaling is essential and sufficient for T cell development, and recently developed in vitro culture systems point to members of the Delta family as being the physiological N1 ligands. We explored the ability of Delta1 (DL1) and DL4 to induce T cell lineage commitment and/or maturation in vitro and in vivo from bone marrow (BM) precursors conditionally gene targeted for N1 and/or N2. In vitro DL1 can trigger T cell lineage commitment via either N1 or N2. N1- or N2-mediated T cell lineage commitment can also occur in the spleen after short-term BM transplantation. However, N2-DL1-mediated signaling does not allow further T cell maturation beyond the CD25(+) stage due to a lack of T cell receptor beta expression. In contrast to DL1, DL4 induces and supports T cell commitment and maturation in vitro and in vivo exclusively via specific interaction with N1. Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak. Interestingly, preferential N1-DL4 binding reflects reduced dependence of this interaction on Lunatic fringe, a glycosyl transferase that generally enhances the avidity of Notch receptors for Delta ligands. Collectively, our results establish a hierarchy of Notch-Delta interactions in which N1-DL4 exhibits the greatest capacity to induce and support T cell development.

Show MeSH
Related in: MedlinePlus